Targeting neurons and photons for optogenetics

Targeting neurons and photons for optogenetics
Nature Neuroscience 16, 805–815 (July 2013)

optogenetic probes

dream experiments will become possible with the application of these new approaches

Caenorhabditis elegans has 302 neurons, and the morphology of every neuron is known.

mammalian retina: the functional unit is often considered to be a mosaic of cells with the same properties, referred to as cell type.

cell type” refers to a population of neurons that cannot practically be divided into smaller units

cell class” refers to a population of neurons that is defined by some common property but which can be further divided into smaller populations.

a single retinal ganglion cell mosaic

Targeting the right neurons is still a largely unsolved problem, especially in species, such as non-human primates, where genetic manipulations are often not feasible.

approaches for targeting optogenetic probes, focusing on using viruses, alone or in combination with transgenics.

the virus used most frequently for targeting, the adeno-associated virus (AAV), has a coat protein that exists in 100 different variants in nature … changing the entry site from axons to soma or dendrites.

The thousands of viruses made by nature and the many variants made by researchers can therefore be thought of as a ‘Legoland’ for neuroscientists performing optogenetics experiments

the way viruses are made is highly modular: the different properties are stored in different plasmids, and by mixing these plasmids and adding them to cells the virus is self-assembled.

Replication-competent viruses are toxic to varying degrees

the genetic identity of these neurons (for example, expression of parvalbumin)

Virus targeting based on genetic identity.
The morphology and function of different cell types is to a large extent defined by the pattern of genes they express.
Past work has used the fact that some classes of neurons uniquely express particular signature genes—for instance, a large class of fast-spiking interneurons expresses parvalbumin

molecular tools, such as site-specific recombinases (for example, Cre or Flp) can be used to drive the expression of optogenetic probes from viruses infecting these cells.
Such conditional viruses can be made from DNA viruses, such as AAV or herpesviruses

The main drawback of the conditional virus approach is that it requires expression of a site-specific recombinase, typically using a transgenic animal.
The generation of a transgenic animal for a target neuronal type is both time consuming and unpredictable

Rabies– and herpesvirus-based retrograde labeling methods, while suitable for short-term studies over days, are too toxic for studies in which long-term expression is needed.

AAVs are excellent tools for anterograde delivery; however, existing AAVs are not exclusively anterograde, and further development of nontoxic, exclusively anterograde vectors is needed.

single-cell electroporation of a postsynaptic neuron and the subsequent initiation of a retrograde virus from only the electroporated neuron.

high-level, long-term expression has been shown to cause abnormal axonal morphology

This involves using two-photon microscopy to target a plasmid-filled patch pipette to individual neurons in vivo, followed by electroporation to deliver the plasmid to the cell under visual control.
Neurons can be targeted in this way on the basis of

  • their somatodendritic morphology (using ‘shadowimaging’30),
  • their genetic identity (using GFP expression as a marker) or
  • their functional properties (such as tuned responses to sensory stimuli) for subsequent optogenetic activation.


Optogenetics: controlling cell function with light
Nature Methods 8, 24–25 (2011)

Optogenetics for Analyzing and Engineering Neural Circuits

Optogenetics for Analyzing and Engineering Neural Circuits
Ed Boyden, PhD
Associate Professor, MIT Media Lab, McGovern Institute Departments of Biological Engineering and Brain and Cognitive Sciences
Case Western Reserve. Mar 29, 2012

Understanding how neural circuits work together to implement brain functions, and how these computations go awry in brain disorders, is a top priority for neuroscience.
Over the last several years we have discovered and developed a rapidly-expanding suite of genetically-encoded reagents (e.g., ChR2, Arch, Mac, ArchT, ChR65, and others) that, when expressed in specific neuron types in the nervous system, enable their activities to be powerfully and precisely activated and silenced in response to pulses of light.
These tools are in widespread use for analyzing the causal role of defined cell types in normal and pathological brain functions.
In this talk I will give a brief overview of these tools, and discuss a number of new tools for neural activation and silencing that we are developing, including new molecules with augmented amplitudes, improved safety profiles, novel color and light-sensitivity capabilities, and unique new capabilities.
We have begun to develop hardware to enable complex and distributed neural circuits to be precisely controlled, and for the network-wide impact of a neural control event to be measured using distributed electrodes and fMRI.
We explore how these tools can be used to enable systematic analysis of neural circuit functions in the fields of emotion, sensation, and movement, and in neurological and psychiatric disorders.

Finally, we discuss our pre- clinical work on translation of such tools to support novel ultraprecise neuromodulation therapies for human patients.

3:20 high-dimensional space cell phenotype space technical mambo-jumbo

14:24 Three major optogenetic molecule classes
A. archaerhodopsins and bacteriorhodopsins (e.g. Arch, Mac, BR)
B. halorhodopsins (e.g. Halo/NpHR)
C. channelrhodopsins (e.g. ChR2) Boyden, E.S. (2011) Faculty of 1000 Biology Reports 3:11

18:44 Lentiviruses and adeno-associated viruses have intrinsic tropism for certain cell types (e.g., lenti-excitatory neurons of the cortex)

Using Light To Tweak The Living Brain

Experimental Tool Uses Light To Tweak The Living Brain
December 26, 2013

a relatively new set of techniques called optogenetics that allows researchers to control the activity of brain cells using light.

“This is fantastic,” says Elizabeth Hillman, a biomedical engineer at Columbia University. “We can turn things on, turn things off, read stuff out.” In short, she says, it provides a way to observe and control what brain circuits are doing in real time in a living brain.

Eventually, optogenetics could not only help explain diseases like epilepsy and depression, but offer a way to treat them. But the technique needs some refinement before it can be used in people or in remote parts of the brain, Hillman says.

in 2005, when a team at Stanford University showed how to control brain cells using light. “There was instant buzz about it,” Hillman says. “People were sort of running around and saying, ‘What is this thing, where can I get it, how can I do it?’ ” she says.

For one thing, Hillman says, when you use optogenetics, “You’re actually altering the genes of the neurons.”
That’s because most neurons don’t normally respond to light.
So you have to add genetic material to every brain cell you want to control. Scientists can do that in mice with genetic engineering, but that’s not an option for people.